Fig 2.

Clr4 is required to establish, but not to maintain CENP-A^Cnp1 and kinetochore proteins on the large cen3 minichromosome. Association of CENP-A^Cnp1 and H3K9me2 in (A), and CENP-C^Cnp3 and Sim4 in (B) were determined by ChIP in wild-type and clr4 strains containing pH-icc3i-H introduced by crossing (X) or transformation (T). fbp1 or act1 are control noncentromeric loci. In CENP-A^Cnp1, CENP-C^Cnp3, and Sim4 ChIPs enrichment of plasmid cc3 product was compared with input DNA (T) relative to the act1 product. Positive control: Enrichment of endogenous centromeric cc1/3 was assessed. H3K9me2 ChIP: Enrichment of plasmid–outer repeat J3 product was compared with PCR of input DNA (T) relative to act1. Positive control: Enrichment of endogenous otr centromeric repeats was assessed.